Cross-talk between G protein-coupled and epidermal growth factor receptors regulates gonadotropin-mediated steroidogenesis in Leydig cells

Abstract

Gonadal steroid production is stimulated by gonadotropin binding to G protein-coupled receptors (GPCRs). Although GPCR-mediated increases in intracellular cAMP are known regulators of steroidogenesis, the roles of other signaling pathways in mediating steroid production are not well characterized. Recent studies suggest that luteinizing hormone (LH) receptor activation leads to trans-activation of epidermal growth factor (EGF) receptors in the testes and ovary. This pathway is critical for LH-induced steroid production in ovarian follicles, probably through matrix metalloproteinase (MMP)-mediated release of EGF receptor (EGFR) binding ectodomains. Here we examined LH and EGF receptor cross-talk in testicular steroidogenesis using mouse MLTC-1 Leydig cells. We demonstrated that, similar to the ovary, trans-activation of the EGF receptor was critical for gonadotropin-induced steroid production in Leydig cells. LH-induced increases in cAMP and cAMP-dependent protein kinase (PKA) activity mediated trans-activation of the EGF receptor and subsequent mitogen-activated protein kinase (MAPK) activation, ultimately leading to StAR phosphorylation and mitochondrial translocation. Steroidogenesis in Leydig cells was unaffected by MMP inhibitors, suggesting that cAMP and PKA trans-activated EGF receptors in an intracellular fashion. Interestingly, although cAMP was always needed for steroidogenesis, the EGFR/MAPK pathway was activated and necessary only for early (30-60 min), but not late (120 min or more), LH-induced steroidogenesis in vitro. In contrast, 36-h EGF receptor inhibition in vivo significantly reduced serum testosterone levels in male mice, demonstrating the physiologic importance of this cross-talk. These results suggest that GPCR-EGF receptor cross-talk is a conserved regulator of gonadotropin-induced steroidogenesis in the gonads, although the mechanisms of EGF receptor trans-activation may vary.

FIGURE 1.

hCG promotes progesterone production in MLTC-1 Leydig cells in a dose- and time-dependent fashion. A, hCG was added to MLTC-1 cells at the indicated concentrations, and progesterone levels in the media were measured after 30 min. B, 5 units/ml hCG were added to MLTC-1 cells, and progesterone levels in the media were measured at the indicated times. Each point/bar represents the mean ± S.D. (n = 3). All studies were performed at least twice with virtually identical results.

FIGURE 2.

EGFR signaling is important for hCG-induced steroidogenesis in Leydig cells. A and B, MLTC-1 mouse Leydig cells were pretreated with 0.1% ethanol, 20 μm AG1478, 20 μm galardin, or 5 μm erlotinib prior to addition of 5 units/ml hCG for 30 min or 2 h. Progesterone content of the media was measured by RIA (A), and cell lysates were examined by Western blot for phosphorylated (B, upper panel) and total (B, lower panel) EGFR. C, 5 units/ml hCG or 20 ng/ml EGF were added to MLTC-1 cells for 30 min or 2 h, and media progesterone content was measured by RIA. D, primary mouse Leydig cells were isolated from sexually mature male mice, placed in 6-well plates, and serum-starved overnight. Cells were then preincubated with 0.1% ethanol or 20 μm AG1478 for 30 min before the addition of 5 IU/ml hCG for 30 min or 2 h. Testosterone content in the media was measured by RIA. For all graphs, each bar represents the mean ± S.D. (n = 3). All studies were performed at least three times with nearly identical results. E, EGF receptor signaling is important for testosterone production in vivo. Seven-week-old male mice were injected with either PBS or PBS containing 20 μm AG1478. Mice were reinjected after 24 h, and serum testosterone levels were measured after a total of 36 h. Each bar represents the mean ± S.E. (n = 11). Using the Student's t test, p = 0.03.

FIGURE 3.

MAPK signaling is necessary for early, but not prolonged, hCG-induced steroidogenesis in Leydig cells. MLTC-1 cells were preincubated with 0.1% ethanol, 20 μm PP2 (A, Src inhibitor), or 20 μm U0126 (B–D, MEK inhibitor), followed by stimulation with 5 IU/ml hCG for the indicated times. Progesterone content in the media was measured by RIA (A and B). MEK activity in cell lysates was determined by Western blot for phosphorylated p42/p44 (C, upper panel) and total p42/p44 (C, lower panel). D, EGFR activation occurs upstream of MEK activation. EGFR activation in cell lysates was determined by Western blot for phosphorylated EGFR (D, upper panel) and total EGFR (D, lower panel). E and F, MEK activation is not inhibited by AG1478 and is not sufficient to promote steroidogenesis. MLTC-1 cells that were pretreated with 0.1% ethanol (Mock) or 20μm AG1478 were incubated with either 5 units/ml hCG, 25 ng/ml FGF-2, or both peptides for 30 min. Media progesterone levels were measured by RIA (F), and cell lysates were analyzed by Western blot for phosphorylated (upper panel) or total (lower panel) p42/p44. F, for all graphs, each bar represents the mean ± S.D. (n = 3). All experiments were performed at least twice with essentially identical results.

FIGURE 4.

cAMP and PKA signaling are necessary and sufficient for hCG-induced activation of the EGFR and MEK, as well as steroidogenesis, in Leydig cells. A–C, MLTC-1 cells were preincubated with 0.1% ethanol (Mock) or 20 μm H-89 (PKA inhibitor), followed by addition of 5 IU/ml hCG for the indicated times. Progesterone content in the medium was measured by RIA (A). Cell lysates were examined by Western blot for phosphorylated and total EGFR (B, upper and lower panels, respectively) and phosphorylated and total p42/p44 (C, upper and lower panels, respectively). D–F, MLTC-1 cells preincubated for 30 min with 0.1% ethanol (Mock), 5 μm erlotinib (EGFR inhibitor), 20 μm U0126 (MEK inhibitor), or 20 μm H-89 (PKA inhibitor), followed by the addition of 10 μm forskolin for the indicated times. Progesterone content in the media was determined by RIA (D). Cell lysates were examined by Western blot for phosphorylation of the EGFR (E) or p42/p44 (F). For all graphs, each bar represents the mean ± S.D. (n = 3). Each experiment was performed at least three times with nearly identical results.

FIGURE 5.

LH receptor-mediated trans-activation of the EGFR regulates early, but not prolonged, phosphorylation and mitochondrial translocation of StAR in Leydig cells. A, MLTC-1 cells were preincubated for 30 min with 0.1% ethanol (Mock) or 20 μm AG1478 (EGFR inhibitor) followed by the addition of 20 μm 22(R)-hydroxycholesterol (Steraloids) for 30 min. Progesterone content in the media was measured by RIA. Each bar represents the mean ± S.D. (n = 3). B, MLTC-1 cells were stimulated with 5 IU of hCG for the indicated times after preincubation with 0.1% ethanol (Mock) or 20 μm AG1478. Mitochondria were isolated and examined by Western blot for total StAR (upper) and phosphorylated StAR. Equal amounts of protein were added to each lane. Each experiment was performed at least three times with essentially identical results.

FIGURE 6.

Model for gonadotropin-mediated steroidogenesis in Leydig cells. Gonadotropin (LH or hCG) activates the LH receptor, leading to a rapid and prolonged increase in cAMP levels and subsequent PKA activity. Early increases in PKA signaling trans-activate the EGFR independent of ligand (left side of figure), leading to activation of the MAPK signaling pathway. By mechanisms that are still not known, MAPK activation leads to phosphorylation and translocation of small levels of existing StAR to the mitochondria, resulting in increased steroidogenesis. Prolonged PKA signaling leads to increased transcription of StAR mRNA, followed by increased StAR protein expression (middle of figure). By 2 h, significant MAPK signaling is no longer occurring nor is it required for StAR phosphorylation and translocation to the mitochondria. Note that LH receptor activation also triggers trans-activation of the EGFR via MMP activation (right side of figure); however, this process is not necessary for steroid production and any time point.